This invention relates to methods for transforming arthropods with exogenous DNA. The methods are useful for arthropods that have not previously been amenable to DNA-mediated transformation, and also provide a simpler and more efficient means of transforming arthropods that have previously been transformed.
Many arthropod species are disease vectors and a far greater number are agriculturally important, including those organisms that are pests, and those that are beneficial. Genetic manipulation of arthropods has the potential to be an extremely important means of altering properties of arthropods to, for example, reduce their ability to spread disease or to increase their effectiveness as biological control agents. It will be possible to modify the temperature tolerance, host range, resistance to insecticides, or sex ratio. Altering these traits could improve the performance of a biological control agent and result in an overall reduction in the use of insecticides.
Classical genetic manipulation of the western predatory mite Metaseizilus occidentalis (Nesbitt), for example, has proven to be highly valuable in using this mite as a biological control agent against spider mites. M. occidentalis is a member of the cosmopolitan family Phytoseiidae, a group of mostly predatory mites responsible for controlling spider mites and other arthropods in many crops (Sabelis, M. W. [1985] In: Spider Mites: Their Biology, Natural Enemies, and Control, eds. Helle, W. and Sabelis, M. W., Elsevier, N.Y., Vol. 1B, pp. 35-40). M. occidentalis is less than 500 .mu.m long (Chant, D. A. [1985], Id. at pp. 3-32), and has five developmental stages: egg, larva, protonymph, deutonymph and adult. At 27.degree. C. the life cycle from egg to gravid female is completed in approximately 6 days. Females lay approximately 2 eggs a day. Only one egg is matured at a time, with the fully mature egg occupying approximately 1/3 of the body cavity. Phytoseiids are thought to have a single unpaired ovary.
Using classical genetic methods, a process that required three years to accomplish, pesticide-resistant M. occidentalis strains were developed previously. These mites were incorporated into an integrated mite managementprogram in California almond orchards and have saved almond growers at least $24-44 per acre each year, primarily by reducing pesticide applications to control spider mites (Headley, J. C., M. A. Hoy [1987] J. Econ. Entomol. 80:555-559)
Genetic improvement of a beneficial arthropod species has thus been shown to be highly effective. Harmful arthropods may also be controlled by genetic manipulation. However, classical genetic techniques have significant drawbacks. Artificial selection may be slow, inefficient, or ineffective. Most genetic improvement projects involve selecting for resistance to pesticides (Hoy, M. A. [1990] In: Pesticide Resistance in Arthropods, eds. Roush, R. T. and B. E. Tabashnik, Chapman and Hall, N.Y., pp. 203-236), but sometimes resistance alleles do not occur in the populations that are being screened. Selection attempts may take more than 2 years, and success is not assured. The use of mutagenesis and hybridization for genetic manipulation remain extremely limited and have significant disadvantages (Beckendorf, S. K., M. A. Hoy [1985] In: Biological Control in Agricultural IPM Systems, Eds. Hoy, M. A. & D. C. Herzog, Academic Press, Orlando, pp. 167-187; Hoy, M. A. [1990], supra.). A more efficient method for developing genetically-improved strains of arthropod biological control agents is needed.
In recent years, substantial research efforts have been directed to the development of direct genetic manipulation techniques which can be utilized to develop plants and animals with useful traits. These techniques can yield highly desirable plants and animals much more quickly and efficiently than through the use of traditional breeding or selection techniques. Bacteria were the focus of much of the early genetic transformation work but, within the last decade, transformation of a wide variety of plants and animals has been attempted. Some of these transformation efforts have been successful (see, for example, Vasil, V., A. M. Castillo, M. E. Fromm, I. K. Vasil [1992] Bio/Technology 10:667-674; Umbeck, P. F., U.S. Pat. No. 5,159,135; Fire, A. [1986] EMBO J. 5(10):2673-2680; Spradling, A., G. Rubin [1982] Science 218:341-347; Rubin, G., A. Spradling [1982] Science 218:348-353), while many of the experiments have been unsuccessful. There remains a great number of organisms for which no method of transformation currently exists.
The development of germ-line transformation in Drosophila melanogaster (Spradling et al., supra) has provided a valuable tool for genetic analysis of this arthropod. Initial efforts to transform arthropods other than Drosophila focused on employing existing P element vectors, in particular pUChsneo (Steller, H., V. Pirrotta [1985] EMBO J. 4:167-171), and microinjecting this vector into preblastoderm eggs (Miller, L. et al. [1987] Science 237:779-781;McGrane, V. et al. [1988] Am J. Trop. Med. Hyg. 39:502-510; Morris, A. C. et al. [1989] Med. Vet. Entomol. 3:1-7). Transformation was achieved occasionally, but at very low frequencies and apparently was independent of P element-mediation. The rare transformation events reported appear to be the result of random integrations. Present evidence indicates the utility of P element vectors is limited in organisms outside the family Drosophilidae(O'Brochta, D. A., A. M. Handler [1988] Proc. Natl. Acad. Sci. USA 85:60532-6056).
Given the current absence of efficient vectors for arthropods other than Drosophila, substantial research has been devoted to developing alternative DNA delivery systems. Investigators have explored ballistic introduction of DNA-coated micro projectiles (Baldarelli, R. M., J. A. Lengyel [1990] Nucl. Acids. Res. 18:5903-5904; Carlson, D. A. et al. [1989] In: Host Regulated Developmental Mechanisms in Vector Arthropods, Borovsky, D. and Spielman, A., eds., Univ. of Florida Press, Vero Beach. Fla., pp. 248-252), electroporation, and puncturing eggs with DNA-coated silicon carbide filaments. Another potential DNA delivery system involves binding heterologous DNA to sperm and introducing it through artificial insemination. This approach may be useful in honey bees (Milne, C. P. [1991] Am. Bee Journal 131:188-189), but it is not known whether the bound DNA can actually be introduced into the embryo by this method.
Aspects of the subject invention have been reported in Presnail, J. K. and M. A. Hoy (1992) Proc. Natl. Acad. Sci. USA 89:7732-7736.